This proposal seeks support for the development of strategies of drug designs which are based on the novel mechanistic properties of enzymes postulated to be the targets in cancer chemotherapy and in the treatment of other diseases. Specifically, during the initial tenure of this grant, we propose to develop a new class of potential mechanism-based inhibitors of glyoxalase I which are "inverse substrates" for this enzyme and rely on our recently discovered mechanistic observations, termed mirror-image catalysis, for their activation to reactive species. We will use some of the assumptions implicit in mirror-image catalysis as a probe for determining the absolute stereochemistry of the glyoxalase I reaction, a question which has eluded solution due to the rapid equilibration of the diastereomeric thiohemiacetals in the normal reaction. The intrinsic stability which may be incorporated into the inverse substrates will permit stereochemical analysis by a number of classical methods. Secondly, the principles of mirror-image catalysis will permit the synthesis of stable, glutathione-containing inverse substrates which may undergo conversion to highly reactive compounds capable of inactivating the enzyme and/or exhibiting cytotoxicity in an in vivo situation at some other site. These compounds will be synthesized by techniques which have been developed in this laboratory. Their mechanistic and kinetic properties will be thoroughly studied with purified glyoxalase I from a number of sources and inhibitory activity will be determined in cell culture. The function of the glyoxalase system has been a point of controversy for nearly seventy years. It has been purported to be merely a detoxification pathway by some researchers to a basic regulatory system of cell growth by others. These studies should shed light on its function and have applicability to the study of other glutathione-dependent processes, such as inflammation and allergic responses.